UNIVERSITY  OF  CALIFORNIA   PUBLICATIONS 

IN 

AGRICULTURAL    SCIENCES 

Vol.  4,  No.  3,  pp.  99-112,  pis.  3-6  February  15,  1919 


ON  THE  EXISTENCE  OF  A  GROWTH-INHIBITING 
SUBSTANCE  IN  THE  CHINESE  LEMON^ 

BY 

H.  S.  EEED  AND  F.  F.  HALMA 


It  is  the  purpose  of  this  preliminary  paper  to  point  out  the  prob- 
able existence  of  a  growth-inhibiting  substance  or  substances  in  the 
stem  of  Chinese  lemon,  a  variety  of  the  citron  {Citrus  medica),  and 
to  discuss  some  of  its  effects  upon  the  development  of  shoots  on  the 
cuttings. 

Vochting"  believed  that  each  isolated  stem  is  the  carrier  of  a 
''force"  which  determines  polarity. 

The  older  botanists,  under  the  leadership  of  Sachs,  assumed  that 
there  was  a  flow  of  shoot-forming  substances  toward  the  apex  and  of 
root-forming  substances  toward  the  base  in  a  regenerating  cutting. 
The  difficulty  in  this  assumption,  so  far  as  the  Chinese  lemon  is  con- 
cerned, lies  in  the  fact  that  none  of  the  subapical  buds  develop.  If 
the  apical  buds  of  the  cutting  develop  as  the  result  of  the  flow  of 
elaborated  materials  in  their  direction,  it  is  difficult  to  see  why  these 
materials  which  flow  past  subapical  buds  should  not  give  them  a 
stimulus  to  develop,  though  less  in  degree. 

In  a  series  of  recent  articles,  Loeb  has  turned  attention  toward 
the  possibility  that  there  are  one  or  more  specific  substances  in  the 
growing  stem  which  determine  the  course  of  events  in  the  growth  of 
new  shoots  and  roots  in  cuttings.  He  has  shown  that  in  Bri/ophylliini 
calycinum  the  apical  bud  prevents  the  lower  ones  from  growing  out, 
and  he  concluded  that  there  is  an  inhibitory  substance  sent  in  the 


1  Paj^er  no.  53,  Univ.  Calif.  Grad.  Sch.  Trop.  Agr.  and  Citrus  Exp.  Sta.,  River- 
side, California. 

2  Yochting,  H.,  Organbildung  im  Pflanzenreich,  Bonn,  1878. 


100  University  of  California  Publications  in  Agricultural  Sciences        [Vol.  4 

direction  of  the  basal  buds.  He^  also  believes  that  the  reason  why  the 
apical  bud  grows  out  first  is  that  it  is  the  first  bud  which  is  freed  from 
this  substance  when  the  stem  is  cut  from  the  mother  plant. 

This  view  receives  strong  support  from  studies  on  the  growth  of 
potato  sprouts  recently  published  by  Appleman.*  He  finds  when  all 
the  buds  on  a  tuber  are  subjected  to  the  same  external  conditions,  that 
the  buds  on  the  apical  end  grow  out  first,  and  that  they  inhibit  the 
growth  of  the  more  basal  buds.  If  these  apical  sprouts  are  destroyed, 
or  retarded  in  their  normal  growth,  sprouts  will  appear  from  the 
more  basal  buds  which  would  otherwise  have  remained  dormant.  The 
cutting  of  a  furrow  around  a  middle  or  basal  bud  will  cause  it  to 
grow  out  just  as  quickl}^  as  the  apical  buds  of  the  tuber.  If  the  tuber 
is  cut  into  transverse  slices  the  inhibitory  influence  of  the  apical  buds 
is  removed  and  there  is  a  general  growth  of  buds  over  the  surface 
of  the  entire  tuber,  depending  upon  the  thickness  of  the  slices. 

In  the  following  experiments  the  cuttings  were  made  from  young 
wood  of  the  Chinese  lemon  and  varied  in  length  from  15  to  40  centi- 
meters. They  were  suspended  in  covered  glass  jars  or  in  glass  cases 
in  which  a  moist  atmosphere  was  continually  maintained.  The  experi- 
ments were  conducted  in  a  culture  room  at  a  temperature  which  had 
a  daily  range  of  not  more  than  one  or  two  degrees  centigrade.  Illumi- 
nation was  obtained  from  two  large  incandescent  electric  lights  fitted 
with  filters  of  "Trutint"  glass.  The  jars  stood  upon  a  revolving  table 
which  was  driven  at  a  speed  of  one  revolution  every  three  minutes  by 
a  small  motor. 

Shoots  of  the  Chinese  lemon  which  have  attained  a  sufficient  degree 
of  maturity  retain  their  vitality  for  many  months  after  removal  from 
the  tree,  provided  they  are  kept  in  a  moist  atmosphere.  The  latency 
of  the  buds  is  not  so  easily  broken  as  in  the  case  of  willow ;  they  there- 
fore constitute  a  more  favorable  subject  for  experimentation. 
Regeneration  of  Shoots  from  Cuttings 

When  cuttings  made  from  shoots  of  the  Chinese  lemon  are  sus- 
pended in  a  moist  atmosphere,  shoots  develop  from  buds  at  the  apex 
of  the  cuttings,  and  roots  from  the  basal  end.  This  occurs  whether 
the  cuttings  are  in  the  normal  or  inverted  position  (figs.  1,  2).  The 
cuttings  will  grow  for  several  months  but  no  shoots  will  appear  except 
from  two  or  three  buds  nearest  the  apical  end  of  the  cutting.    If  the 

3  Loeb,  J.,  The  chemical  basis  of  axial  polarity  in  regeneration,  Science,  n.s., 
vol.  46,  p.  547,  1917. 

4  Appleman,  C.  O.,  Physiolofjical  basis  for  the  preparation  of  potatoes  for 
seed,  Bull.  212,  Maryland  Agr.  Exp.  Sta.,  1918. 


1919]       Eeed-Halma:  Growth-Inhibiting  Substance  in  the  Chinese  Lemon  101 

buds  nearest  the  apex  are  killed  by  burning  them  with  a  hot  glass  rod 
at  the  time  of  installing  the  experiment  then  the  buds  immediately 
below  them  will  develop,  but  if  the  apical  buds  remain  functional  and 
develop  the  lower  buds  remain  dormant.  If  the  sprout  from  an 
apical  bud  be  removed  after  it  has  reached  a  length  of  one  centimeter 
or  more,  a  new  sprout  will  develop  from  one  of  the  accessory  buds 
but  the  lower  buds  remain  dormant.  If  the  tip  of  a  sprout  be  removed 
as  in  the  left-hand  cutting  in  figure  1,  it  will  continue  to  grow  from 
one  of  its  lateral  buds  but  the  interruption  of  its  growth  does  not  cause 
the  buds  below  it  to  develop. 

Similar  behavior  is  shown  by  shoots  on  the  Chinese  lemon  tree 
itself.  Figure  3  shows  four  upright  shoots,  averaging  one  meter  high, 
which  were  taken  from  a  Chinese  lemon  tree.  A  few  lateral  buds  near 
the  apex  of  each  shoot  had  developed  into  short  branches,  yet  the 
large  number  of  buds  below  them  had  remained  dormant. 

This  dominance  of  the  buds  nearest  the  apex  of  the  shoots  is  so 
characteristic  and  so  clearly  marked  that  one  can  only  conclude  that 
it  is  due  to  some  significant  internal  influence. 

We  may  now  turn  our  attention  to  some  experiments  designed  to 
throw  light  upon  the  nature  of  this  influence. 

Cuttings  about  30  centimeters  in  length  were  suspended  vertically 
in  a  glass  jar  until  one  or  two  shoots  were  produced  at  the  apex.  A 
notch  deep  enough  to  remove  bark  and  phloem  was  then  cut  immedi- 
ately above  several  subapical  buds  and  the  cuttings  replaced  in  the 
jar.  After  three  weeks  nearly  every  healthy  bud  above  which  a  notch 
had  been  made  either  produced  a  shoot  or  showed  signs  of  activity 
(fig.  1).  The  unnotched  buds  remained  dormant.  The  same  result 
was  obtained  when  the  cuttings  were  hung  upside  down  (fig.  2) .  None 
of  the  buds  on  the  control  cuttings  developed  except  those  immediately 
back  of  the  apex.  The  response  is  often  more  manifest  if  the  sprout 
from  the  apical  bud  is  allowed  to  reach  a  length  of  one  or  two  centi- 
meters before  notching  the  subapical  buds. 

The  next  experiment  shows  what  happens  when  the  development 
of  the  apical  buds  is  temporarily  inhibited  by  mechanical  means. 

Cuttings  about  30  centimeters  in  length,  of  as  uniform  size  as 
possible,  were  selected  and  the  upper  half  of  some  of  them  was  enclosed 
in  a  plaster  of  Paris  cast.  They  were  then  suspended  vertically  in  a 
glass  case  and  placed  on  the  revolving  table.  In  most  cases  the  casts 
prevented  development  of  the  buds.  After  a  little  over  three  weeks 
some    of   the    cuttings   produced    a   sprout   below   the    plaster    cast. 


102  University  of  California  Publications  in  Agricultural  Sciences        [Vol.  4 

Figure  5  shows  two  of  these  cuttings  on  which  the  new  sprouts  were 
8  and  11  centimeters  in  length  respectively.  The  sprout  on  each 
cutting  was  allowed  to  grow  until  it  reached  a  length  of  several  centi- 
meters, then  the  plaster  cast  was  removed  and  the  cutting  again 
suspended  in  the  moist  chamber. 

The  behavior  of  the  cuttings  released  from  the  plaster  cast  was 
significant.  Soon  after  removing  the  casts  the  apical  buds  of  each 
cutting  developed  into  sprouts  in  the  normal  manner.  Figure  6  is 
from  a  photograph  of  the  two  encased  cuttings  shown  in  figure  5, 
and  was  taken  one  month  after  removing  the  casts.  It  shows  that  the 
shoots,  which  had  previously  developed,  began  to  die  when  the  apical 
buds  began  to  grow.  Subsequently  the  original  shoots  died,  while 
those  from  the  apical  buds  grew  on  normally.  The  result  of  this 
experiment  shows  two  significant  facts:  (1)  the  shoots  produced  from 
apical  buds  after  removing  the  casts  were  not  inhibited  in  any  manner 
by  those  already  present  near  the  middle  of  the  cuttings;  and  (2),  on 
the  contrary,  the  original  shoots  near  the  middle  of  the  cuttings  were 
inhibited  in  their  growth  and  finally  killed  when  the  apical  shoots 
began  to  grow.  The  length  of  the  sprout  produced  by  the  subapical 
bud  might  alter  these  relations,  for  example  Mogk^  reported  that 
subapical  sprouts  in  Vicia  faha  seedlings  did  not  inhibit  growth  of 
the  apical  sprouts  unless  their  relative  lengths  were  as  three  to  one. 

The  results  of  these  experiments  seem  to  us  to  indicate  that  there 
is  some  substance  produced  by  the  growing  shoot  which  travels  in  the 
phloem  layers  toward  the  basal  part  of  the  stem  and  that  it  inhibits 
the  development  of  buds  situated  nearer  the  base  of  the  stem.  In 
making  a  cutting  such  as  that  shown  at  the  left  in  figure  1,  we  are 
isolating  a  piece  of  a  stem  whose  buds,  according  to  this  assumption 
would  have  been  previously  prevented  from  developing  by  a  substance 
emanating  from  the  upper  shoots  or  apical  buds.  If  the  substance 
which  was  included  in  this  cutting  remained  evenly  distributed 
throughout  its  length,  none  of  the  buds  would  have  developed,  but  its 
tendency  seems  to  be  to  migrate  toward  the  basal  end  of  the  cutting. 

The  question  might  arise  whether  the  results  may  not  be  due  to  a 
tendency  for  elaborated  materials  to  pass  upward  more  than  down- 
ward, and  to  induce  the  development  of  apical  buds  only,  through  an 
accumulation  of  nutrients  at  the  apical  end.  This  question  seems  to 
be  answered  in  the  negative  by  two  observations:  (1)  so  far  as  known, 
tlie  movemcMt  of  su])stan(H3  in  the  phloem  is  downward;  (2)  the  shoots 


•>  Moj^U,  W.,  Tlntersuchunf^cn   iiber  Korrelation   von   Knospen   und   Sprossen, 
Arfh.  f.  KntvvickelHmoch.  d.  Org.,  vol.  38,  pp.  584-681,  1914. 


1919]       Beed-Ralma:  Groivth-Inhibiting  Substance  in  the  Chinese  Lemon  103 

produced  from  short  cuttings  having  only  one  or  two  buds  were  as 
large  and  grew  as  fast  as  those  produced  from  the  first  or  second 
apical  bud  of  a  longer  cutting. 

It  cannot  be  assumed,  however,  that  the  movement  of  this  hypo- 
thetical substance  is  directed  solely  by  the  pull  of  gravity.  As  shown 
by  the  grow^th  of  cuttings  such  as  illustrated  in  figure  2,  it  moves 
from  apex  to  base  even  when  the  cutting  is  suspended  inverted,  in  a 
vertical  position.  It  would  appear  that  the  substance  moves  primarily 
in  a  basal  direction,  but  that  it  is  influenced  to  some  extent  by  the 
pull  of  gravity. 

The  formation  of  new  shoots  on  horizontal  branches  or  on  hor- 
izontally placed  cuttings  of  Chinese  lemon,  affords  some  significant 
illustrations  supporting  the  assumptions  concerning  the  ex:istence  of 
a  growth-inhibiting  substance.  The  horizontal  shoot  on  a  tree  behaves 
very  differently  from  vertical  shoots,  such  as  those  shown  in  figure  3. 
Whereas  very  few  lateral  buds  develop  on  a  vertical  shoot,  a  great 
many  buds  develop  on  a  horizontal  shoot,  provided  they  have  reached 
a  sufficient  degree  of  maturity.  Figure  4  shows  a  long  shoot  which 
originally  grew  vertically  on  the  tree.  So  long  as  it  was  vertical  none 
of  the  lateral  buds  developed  into  shoots.  It  was  bent  over  and  tied 
in  a  horizontal  position.  A  few  months  later  this  photograph  was 
taken  and  shows  a  development  of  lateral  buds  along  nearly  the 
entire  length  of  the  shoot.  Shoots  of  the  same  age  left  in  the  vertical 
position  remained  like  those  shown  in  figure  3.  Inspection  of  the 
branch  will  show,  furthermore,  that  the  buds  which  developed  are 
those  situated  on  the  dorsal  or  upper  side.  Buds  located  on  the  lower 
side  of  the  horizontal  shoot  did"  not  develop.  This  was  true  of 
branches  which  grew  naturally  in  a  horizontal  position,  as  well  as  of 
those  which  were  bent  into  that  position. 

Cuttings  suspended  horizontally  in  glass  cases  produced  new 
sprouts  only  from  buds  on  the  upper  side,  but  not  from  the  lower 
side  (fig.  7).  The  sprouts  were  not  confined  to  the  apical  end  of  the 
cutting,  though  they  usually  appeared  first  in  that  region.  The  growth 
of  sprouts  at  the  apical  end  exhibited  no  such  signs  of  dominance  over 
the  other  buds  on  the  upper  side  of  the  cutting  as  was  shown  in  the 
case  of  cuttings  suspended  vertically. 

This  development  of  the  dorsal  buds  of  a  horizontal  Chinese  lemon 
shoot  appears  to  be  in  conformity  with  the  idea  of  a  growth-inhibiting 
substance  in  the  shoot.  It  appears  that  the  substance  accumulates  on 
the  lower  side  of  the  shoot  and  prevents  its  buds  from  developing, 


104  University  of  California  Publications  in  Agricultural  Sciences        [Vol.  4 

at  the  same  time  freeing  the  upper  side  and  allowing  buds  on  that  side 
to  develop  into  new  shoots. 

A  very  striking  result  was  obtained  if  a  horizontal  cutting  was 
revolved  through  an  arc  of  180  degrees,  so  that  the  position  of  the 
upper  and  lower  buds  was  reversed,  after  sprouts  on  the  dorsal  side 
had  attained  a  length  of  6  to  10  centimeters.  A  new  sprout  would 
soon  appear  from  a  bud  upon  what  was  previously  the  ventral  side 
of  the  cutting.  As  soon  as  this  new  sprout  began  to  grow,  the  original 
sprouts  began  to  deteriorate  and  finally  died  (fig.  8). 

It  seems  possible  that  the  injury  to  the  original  sprouts  might  have 
been  due  both  to  the  growth-inhibiting  substance  originally  present  in 
the  cutting,  and  to  additional  material  produced  by  the  new  sprouts. 
At  any  rate  this  substance  appeared  to  accumulate  at  the  lower  side 
of  the  cutting  and  to  inhibit  growth  in  the  shoots  which  had  been 
brought  into  that  position  by  the  revolution  of  the  cutting.  It  is 
important  to  notice  that  the  two  original  sprouts  on  this  cutting  did 
not  respond  normally  to  the  geotropic  stimulus  previous  to  their 
death.  This  would  indicate  that  there  were  profound  changes  in  the 
metabolism  and  in  growth  reactions  of  the  sprouts. 

It  should  also  be  noticed  that  the  root  which  developed  on  the 
cutting  shown  in  figure  8  showed  no  injury  following  the  revolution 
of  the  cutting.  It  responded  to  its  new  position  by  curving  and  grow- 
ing downward  as  before.  The  influence  of  this  hypothetical  substance 
upon  root  development  is  under  investigation  and  will  not  be  discussed 
here. 

Summary 

1.  Chinese  lemon  shoots  produce  very  few  lateral  branches  so  long 
as  they  grow  in  a  vertical  position.  Cuttings  produce  two  or  three 
new  shoots  from  buds  nearest  their  morphologically  apical  end.  This 
dominance  of  growing  buds  at  the  apex  of  a  shoot  is  characteristic  and 
is  also  shown  by  inverted  cuttings. 

2.  The  dominant  influence  of  the  buds  nearest  the  apex  may  be 
prevented  from  reaching  lower  buds  by  notching  the  phloem  layers 
just  above  each  bud.  If  the  apical  bud  is  prevented  from  developing 
by  mechanical  means,  lower  buds  may  develop. 

3.  Horizontal  branches  or  cuttings  of  this  tree  produce  lateral 
shoots  only  from  the  dorsal  or  upper  side. 

4.  The  theory  is  advanced  that  the  shoots  developing  nearest  the 
apex  form  a  substance  which  is  capable  of  inhibiting  the  growth  of 


1919]      Eeed-Halma:  Growth-Inhibiting  Substance  in  the  Chinese  Lemon  105 

other  buds  on  the  vertical  stem.  The  hypothetical  substance  appears 
to  move  toward  the  morphologically  basal  end  of  a  vertical  shoot  or 
piece  of  shoot.  It  appears  to  have  a  strong  deleterious  effect  upon 
growth  and  to  perpetuate  a  condition  of  dormancy  in  subapical  buds. 
In  horizontally  placed  shoots  this  substance  appears  to  settle  to  the 
lower  side  of  the  shoot. 


PLATE  3 

Fig.  1.  Cuttings  of  Chinese  lemon  which  had  been  suspended  vertically. 
Cutting  at  the  left  produced  two  new  shoots  from  the  buds  nearest  the  apex. 
Cutting  at  the  right  M^as  notched  just  above  several  buds,  some  of  which 
developed  into  new  shoots. 

Fig.  2.  Cuttings  of  Chinese  lemon,  inverted  and  suspended  vertically. 
Cutting  at  the  left  produced  new  shoots  from  buds  nearest  the  apical  end  of  the 
cutting.  Cutting  at  the  right  was  notched  by  removing  bits  of  bark  and  phloem 
just  above  the  buds.    Most  of  the  notched  buds  developed  into  new  shoots. 


[100] 


oro." 


PLATE  4 

Fig.  3.  Vertical  shoots  taken  from  a  Chinese  lemon  tree.  Each  shoot  has  a 
few  small  branches  shortly  below  the  apex,  but  no  others. 

Fig.  4.  Branch  of  a  Chinese  lemon  tree  which  was  bent  horizontally  from 
an  originally  vertical  position.  Buds  on  the  upper  side  of  the  branch  developed 
into  shoots. 


[108] 


UNIV.    CALIF.    PUBL.    AGR.    SCI.,    VOL.    4 


[REED-HALMA]    PLATE    4 


Fio-. 


Fio-.  4 


PLATE  5 

Fig.  5.  Cuttings  of  Chinese  lemon  suspended  vertically.  The  apical  end  of 
two  cuttings  was  enclosed  in  a  plaster  cast.  In  each  case  a  shoot  was  produced 
from  the  first  healthy  bud  below  the  cast.     Control  cutting  shown  at  the  left. 

Fig.  6.  The  two  cuttings  shown  at  the  right  of  figure  5.  Photograph  made 
one  month  after  removal  of  the  casts.  The  subapical  shoots  shown  in  figure  5 
died  after  new  shoots  developed  from  the  apical  buds. 


[110] 


ore 


PLATE  6 

Fig.  7.  Cuttings  of  Chinese  lemon  suspended  horizontally.  Buds  on  the  upper 
side  developed  into  shoots. 

Fig.  8,  Cutting  of  Chinese  lemon  suspended  horizontally.  After  it  had  pro- 
duced two  new  shoots  from  the  upper  side  the  cutting  was  revolved  through 
an  arc  of  180°,  so  that  the  position  of  the  upper  and  lower  buds  was  reversed. 
A  new  shoot  was  produced  from  what  was  then  the  upper  side  and  the  two 
original  shoots  died. 


[112] 


UNIV.    CALIF.    PUBL.    AGR.    SCI.,    VOL.    4 


[REED-HALMAj    PLATE    6 


Fig. 


^^^^^^H^^H 

Fio,  8 


